---start immuno 1.7.97--- next week we'll be doing hypersensitivity rxns. four handouts were given for us to go over in preparation. also a good idea to go over the book. also, the schedule is ending up with three lectures in the last week, which are very detailed and extensive. but most of what we're going to cover from here on out is basically applied immunology, where the concepts we've already learned come back into play in different mechanisms. hopefully we'll eventually figureout how all these cytokines et al work wrt the immune system. today: transplantation: not routinely done in vet med except experimentally, but part of basic medical education. terminology: autograft: moving a part of one body to another part of same body (eg skin) isograft: moving a part from one body to another genetically identical body (eg between monozygotic twins or between inbred animals. allograft: moving a part from one body to another totally unrelated body of the same species xenograft: moving a part from one body to another body of a different species. this is being tried using pigs these days. if you have two inbred strains of animals a and b from a to a transplants are accepted from b to a transplant is rejected from b to ab f1 offspring is accepted from ab f1 to b parent is rejected you always worry about recipient seeing non-self antigen. skin graft to allogeneic individual: graft rejected 10-11 days. this is a first set rejection which occurs with histoincompatibility. skin graft to minor antigen incompatible individual, rejected over about two mos. when you do incompatible allogenic graft: looks fine to five days, then at about 12 days turns black and dies. this first set rejection is primarily mediated by T cells, and TH cells and CD4+ cells. if you let this animal go on, and after a few mos time transplant the SAME donor tissue in, you get a second set rejection at about day 7, graft turns white. this happens much faster than the first rejection due to presence of memory cells and humoral antibody involvement. the longer an individual is exposed to histoincompatible ag, the more the humoral immunity kicks in. but the T cells still mediate most of the rejection. whiteness is due to lack of vascularization, mainly. speed of rejection rxns acute: T cells, primary activation accelerated: reactivation of sensitized t cells chronic: whole system: ab, immune complexes, cellular rxn, etc. hyperacute: minutes/hours if you graft a kidney to an individual already sensitized to donor blood group, graft is rejected as a white graft, occurs in minutes to hours, and is due to fact that you get destruction of blood vessels in grafted kidney via Ab rxn w/endothelium, complement fixation, thrombosis, mac formation, etc. this is hyperacute rxn. HOW does the recipient get imm resp triggered? what does recip see so far as antigen? skin graft. in normal skin there are langerhans cells which express MHCII. they migrate to recip LN where they meet recip T cells. recip CD4+ cells recognize the foreign MHCII on the donor dendritic cells. as many as 5-8% of the T cells will recognize and respond to this. now, when you do a transplant, in the process of doing it, a few of the donor tissue cells are going to die. they will disintegrate and be phagocytosed by recipient macrophages. these macrophages will process them and present antigen to the T cells. so some of the foreign MHCII through processing will get expressed on surfaces of recip macrophages, further stimulating T cells. so t cell stim is both direct and indirect. so T cells clonallyl expand, go through efferent lymphatics, ducts, circ system, and get to grafted tissue, and attack via their mechanisms previously described. so this is how graft gets rejected. the amazing part is the huge # of T cells which can directly recognize foreign MHC - this is an order of magnitude larger than one might expect for any other antigen, so you get a huge ass response. ok. MHCI and MHCII on slide with peptides in clefts. recall that MHCI only holds small peptides while MHCII has an open groove to hold larger molecules. NOW, AA composition of MHCI or MHCII is what the recipient is recognizing. the assumption is that most of MHCI is holding a peptide in the groove, and the peptide could be either a donor or recip peptide. and so the recip recognizes both the peptide and the mhc, but MOSTLY the MHC AA composition. donor cells - epi cells - express MHCI. when they go into recip body, recip T cell notices foreign MHC. ALSO, donor APC has donor MHCI and II and that is recognized by recip TH CD4+ cells. the TH cells will make cytokines: IL2, IFNg (made by TH1) and this will stimulate the cytotoxic CD8+ t cells which will directly contact foreign cells and kill them... also IL2 is needed for the TH cells to expand. further,the TH2 cells' IL2, IL4 and IL5 stimulate the B cells which will make antibody against donor endothelial tissue, leading to destruction of donor blood vessels.. IFNg and TNFb also activate macrophages. when activated macrophages get into graft they cause intense inflammatory response and destruction of graft. first set rejection mostly mediated by T cells and macrophages. the longer the graft persists, you start getting B cells, Ab, and complement involved... realize that these mechanisms are not all 100% one way or the other. there is a mixture, and an interplay of the various mechanisms,and they influence eachother in various ways. ROle of T cells in graft rejection week 0, thymectomy (mice still have T cells - thymus is removed so no NEW t cells can be made) week 3 and 4: injection w/Ab against CD4 (wipes out helper cells). another group against CD8. (wipes out cytotoxic ts) control group no injections week 12: allogenic skin graft week 27 assess graft survival so,which mice should have successful grafts? which monoclonal Ab would be expected to more significantly prolong the graft success? mice who got anti-CD4 Ab have greatly prolonged graft survival. anti cd8 mice very close to control mice. there's no way of getting IL2 or gamma IFN without CD4 cells, so you really don't get a cell mediated response. CD8+ cells cna't by themselves cause massive rejection because if htey did the graft would have been rejected in this experiment.... what part of MHCI in terms of AA composition is really seen as an Ag by recip? and MHCII? well, recall the several alpha domains and one beta2 microglobulin of MHCI. the beta2 is only there to hold up the rest of the molecule on the cell, so it doesn't fall over or something. the main antigenicity seems to reside in the alpha one part of the MHCI,and to some extent the alpha 2 domain as well. in MHCII, the major antigenicity resides in the beta chain, not the alpha chain. -------break------ slide: MHC gene layout. we should note that all the genes in the locus get inherited as a BLOCK. there is not very much crossover. in humans the classone molecules are A B and C. the main problem is associated with molecules at te A and B loci. C genes aren't too much of a problem with rejection etc. also in humans class two are DP DQ DR and DR is the one that we type for because it's most involved in histocompatibility problems. class one mols are best detected serologically, they are serologically defined antigens. class two are best detected in cell to cell interaction eg the are leukocyte defined antigens. so to test for compatibility, best to test using cell to cell interacting. in hmans we call this HLA, cats FLA, cows BLA: (species) leukocyte antigen system. what's important is they are inherited as a block. so you have two parents each with two loci. that's four total. kids end up haploidentical (?) each having one from each parent. note that some minor histocompatibility antigens are not inherited this way thoguh so siblings can have long term rejection problems. so if you have materna andpaternal inheritance, on the cell surface you get both expressed on surface. you get a couple million expressed on the cell surface, actually. each locus has possibility of having several alleles, and offspring get one allele from each parent, so the list gets rediculously long. the amount of variability is enourmous and it is considered that only about two people in 300000 are closely matched with one another in a random population. if you go to countries in europe where there is more *ahem* not inbreeding, but close association between folks in their own villages, there is a lot more compatibility and expression of one allele or another, because *ahem* they knew eachother a little better. but in the us, with all this "interbreeding" from people of widely divergent backgrounds, it's harder to get a match. (he said this not me, don't go calling me names) [i can't help wondering: how much of this do we need to know?] the way people have figured this outis by testing with serum. where did serum come from? primarily from women who have at least 3-4 kids, because their sera has antibody to paternal Ag that were carried by fetus. now,why doesn't this cause fetal rejection? because these antibodies do not cross the placenta. also, people who already have one transplant...who are starting to reject organ. also, transfusion recipients may make Ab to foreign MHC on leukocytes. first, test for blood group antigens - if you do a transplant you need to have identical blood groups. then, microcytotoxicity assay then if you have time, a mixed leukocyte culture test/mixed lymphocyte interaction test. they take the buffy coat, put wbc into wells, also take them from donor recip: mhc a/a donor: mhc d/d treat cells with anti-a, anti-b, add rabbit complement... note we don't even know all the alleles. here we find no match, because after we follow these steps, everything is killed, so it is a total mismatch. rabbits, btw, have naturally occuring ab against human cell surface Ag. that's why we use rabbit complement. [oh god this is dry stuff] so. if a cell gets killed it is labelled with dye and if it turns red it has been killed. viable cells can keep dye OUT of the cytoplasm, but dead cells can't. so you count the eosinophilic cells and you can see if there has been cytotoxicity or not. this is not done by machine, it uses highly trained technologists. if you're getting a heart or kidney, wouldn't it be nice to be tested for MHCII? yes. but, you don't have time to do the mixed lymphocyte culture test, it takes about seven days. you mix lymphs from recip and from donor in culture system and let them react. the recip cells will grow out and proliferate over time and you can measure the proliferation. you're interseted in finding out if recip responds against donor. donor cells are treated with mitomycin c which will block them from proliferation - they can act only as stimulator cells. so they are viable but nondividing cells. so, the more proliferation you get, the more incompatibility there is. so, who's going to do the main triggering here? the B cells and macrophages of the donor. and the main responder is the T cells of the recipients. so this is a good test to do if you have time, but you don't usually. tripp didn't understand how this would be useful...given the 2:300000 ratio of matches. instructor explained how immunosuppressive drugs are used. if you have a match at all loci, no proliferation. if you have a match of MCHI only loci, massive proliferation, no cytotoxicity if other way, massive cytotoxicity, minimal proliferation both don't match: massive proliferation and cytotoxicity immunogenicity of different tissues: from most to least: bone marrow skin islets of langerhans heart kidney liver why? bone marrow and skin contain a lot of monocyte/macrophage/langerhans cells all expressing mhcII. but heart and kidney, esp after flusing out blood, don't have much MHCII at all. there aren't many donor macrophages in those organs, see. corneal transplantation: you can donate a cornea to almost anyone: they aren't vascular, and they have no APCs. if you transplant an avascular cornea, 85% still clear after a year. BUT if you start w/vascular cornea or it becomes vascular while healing, only 33% of those last a year. so if vascularized, try matching, but if not, it's not useful. if becomes vascularized, tx w/topical steroids immunosuppression w/drugs: a number of drugs have really advanced transplantation significantly. steroids: antiinflammatory and mildlyimmunosuppressive. since graft rejection is inflammatory this is helpful. blocks IFNg getting to APC cells, makes membranes more rigid, inflammatory resp becomes much slower, macrophages have more rigid membranes and poorer processing and are less destructive. cyclosporin: a cyclic peptide from a fungus. can treat recipient and it will block at the gene level transcription for IL2 and IFNg so you don't get expansion of T cells, you don't trigger cytotoxic T cells- but it's renal toxic and very immunosuppressive so makes recip prone to secondary infxns. also, after a while, on these drugs, you are prone to b cell lymphoma. azathioprine: blocks proliferation of cytotoxic and other cells. mainly blocks all proliferation of Tc and Th cells. since all t cells have CD3, you coulld make monoclonal Ab to that, you could also target CD4, you could do many monoclonal things...but this is very expensive, and has to be made in different species, so eventually recip starts making antibody to the antibody. you could couple a toxin to an antibody directed against a histocompatibility ag, and you could knock out t cells with that Ab, and you could try other things, but something they're trying now in piglets, is to engineer them so they express on their cell surfaces increased amounts of DAF/MCP, CD59 - the protective molecules that inhibit complement activation - much rxn would be at antibody level in a xenograft, so having these protective molecules could be very helpful in protecting the donor organ. GRAFT vs HOST disease (marrow transplant: often needed for chemo patients, etc. but you need to worry about the donor marrow attacking the patient! you're transfering immunocompetent cells into the immunosuppressed recipient host) donor A donates immunocompetent cells (eg, bone marrow transplant) to x-irradiated recipients C and AB. donor cells divide and attack the foreign antigens - kills mice. in humans causes big spleens, big LNs, GI problems, and death. this occurs because of immunocompetent donor cells responding to recip histocompatibility antigen. now, you NEED immunocompetent cells from donor to trigger this rxn. BUT.most of destruction going on in the tissues are actually caused by HOST cells that are recruited by the cytokines secreted by the donor cells. but you need the trigger of the donor immunocompetent cells. in chick embryos it was shown that this is the case... you can take 14 day old chick embryo, and you cut a window into eggshell, and give IV histoincompatible T cells, you get huge splenomegaly, and then you look at spleen and find that in first three days main proliferating cels are donor cells, but by day 7 it is almost exclusively host cells that are dividing and necrosing in the spleen. so donor cells initiate, but main damaging response is done by host cells. read the book transplant chapter...also know th1 and 2 pos and neg selection in thymus how Ag processed in diff cells (MHCI and II) ----end----